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Abstract
Hypoxia-inducible factors (HIFs) are the master regulators of angiogenesis, a process that is impaired in patients with diabetes mellitus (DM). The transcription factor aryl hydrocarbon receptor nuclear translocator (ARNT, also known as HIF1β) has been implicated in the development and progression of diabetes. Angiogenesis is driven primarily by endothelial cells (ECs), but both global and EC-specific loss of ARNT-cause are associated with embryonic lethality. Thus, we conducted experiments in a line of mice carrying an inducible, EC-specific ARNT-knockout mutation (ArntΔ EC, ERT2) to determine whether aberrations in ARNT expression might contribute to the vascular deficiencies associated with diabetes. Mice were first fed with a high-fat diet to induce diabetes. ArntΔ EC, ERT2 mice were then adminstrated with oral tamoxifen to disrupt Arnt and peripheral angiogenesis was evaluated by using laser-Doppler perfusion imaging to monitor blood flow after hindlimb ischemia. The ArntΔ EC, ERT2 mice had impaired blood flow recovery under both non-diabetic and diabetic conditions, but the degree of impairment was greater in diabetic animals. In addition, siRNA-mediated knockdown of ARNT activity reduced measurements of tube formation, and cell viability in human umbilical vein endothelial cells (HUVECs) cultured under high-glucose conditions. The ArntΔ EC, ERT2 mutation also reduced measures of cell viability, while increasing the production of reactive oxygen species (ROS) in microvascular endothelial cells (MVECs) isolated from mouse skeletal muscle, and the viability of ArntΔ EC, ERT2 MVECs under high-glucose concentrations increased when the cells were treated with an ROS inhibitor. Collectively, these observations suggest that declines in endothelial ARNT expression contribute to the suppressed angiogenic phenotype in diabetic mice, and that the cytoprotective effect of ARNT expression in ECs is at least partially mediated by declines in ROS production.
Highlights
Diabetes mellitus (DM) is one of the leading causes of death in the United States and is frequently associated with cardiovascular abnormalities (Hadi and Suwaidi, 2007; Severino et al, 2018; Knapp et al, 2019) that can lead to critical limb ischemia and amputation (Falanga, 2005)
We were not able to detect a significant reduction in ARNT mRNA in response to high glucose (HG); cell culture with prolonged HG exposure caused a decrease of Arnt mRNA in human umbilical vein endothelial cells (HUVECs) (Supplementary Figure 1)
The mutant line was generated by breeding Arntflox/flox mice in which exon 6 of Arnt is flanked by loxP sequences (Wu et al, 2014), with VE-cadherin-CreERT2 mice expression of Cre-recombinase is tamoxifen-inducible and regulated by the vascular endothelial (VE) Cadherin promoter (Han et al, 2014; Okabe et al, 2014; Figure 1C)
Summary
Diabetes mellitus (DM) is one of the leading causes of death in the United States and is frequently associated with cardiovascular abnormalities (Hadi and Suwaidi, 2007; Severino et al, 2018; Knapp et al, 2019) that can lead to critical limb ischemia and amputation (Falanga, 2005). ARNT is a member of the basic helix-loop-helix/PerARNT-SIM (bHLH/PAS) nuclear receptor family (Madan et al, 2002) and regulates the expression of genes involved in cell survival, angiogenesis, and glucose metabolism by dimerizing with HIF1α under hypoxic conditions. Arnt deficiencies in mice lead to angiogenic defects and embryonic lethality, while Arnt−/− embryonic stem cells fail to respond to declines in glucose concentration (Maltepe et al, 1997). Given that endothelial-specific ARNT mutations are generally associated with embryonic lethality (Maltepe et al, 1997), we characterized the angiogenic role of endothelial ARNT expression under diabetic conditions by conducting experiments in mice carrying an inducible, ECspecific ARNT-knockout mutation. Our results indicate that the angiogenic response to peripheral ischemic injury is impaired in diabetic mice and exacerbated by deficiencies in EC ARNT expression. ARNT inactivation is associated with declines in the angiogenic activity of endothelial cells (ECs) when the cells are cultured under highglucose conditions
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